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Application of clonorchis sinensis recombination protein CsHscB to cholestasis-induced liver fibrosis therapeutic drugs

A clonorchis sinensis, recombinant protein technology, applied in the direction of drug combination, peptide/protein composition, recombinant DNA technology, etc., can solve the problem of unclear mechanism of action, and achieve the effect of significant therapeutic effect

Active Publication Date: 2019-09-17
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Clonorchis sinensis CsHscB protein, as a member of the Hsp family, plays an important role in the immune regulation of Clonorchis sinensis infection and the development of pathological damage is still unclear.

Method used

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  • Application of clonorchis sinensis recombination protein CsHscB to cholestasis-induced liver fibrosis therapeutic drugs
  • Application of clonorchis sinensis recombination protein CsHscB to cholestasis-induced liver fibrosis therapeutic drugs
  • Application of clonorchis sinensis recombination protein CsHscB to cholestasis-induced liver fibrosis therapeutic drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, the preparation of Clonorchis sinensis recombinant protein CsHscB

[0045] 1. Construction of recombinant expression vector

[0046] Primer design

[0047] Referring to the gene sequence of Clonorchis sinensis Henan strain (GenBank: DF143487.1), specific primers were designed using Primer premier 5 software to amplify the entire sequence of CsHscB protein coding gene of Clonorchis sinensis.

[0048] The restriction endonuclease BamHI cutting site (GGATCC) was introduced into the upstream primer P1, and the sequence of the upstream primer P1 was as follows: 5'-CAGCAAATGGGTCGCGGATCCATGTCATTTCGTCTCGCACCA-3'.

[0049] A restriction endonuclease XhoI cutting site (CTCGAG) was introduced into the downstream primer P2, and the sequence of the downstream primer P2 was as follows: 5'-GTGGTGGTGGTGGTGCTCGAGTTAATGCGGAGGAATATCAACTGT-3'.

[0050] Both the upstream primer P1 and the downstream primer P2 mentioned above were synthesized by Shanghai Biological Biotechnol...

Embodiment 2

[0168] Example 2: rCsHscB improves DDC-induced cholestatic liver fibrosis

[0169] 1. Establishment and grouping of animal models of cholestatic liver fibrosis

[0170] Experimental animals: C57BL / 6 mice used in the experiment were purchased from Beijing Speifu Biotechnology Co., Ltd., female, 6-8 weeks old, weighing 20-30 g, SPF grade, and placed in Xuzhou Medical University SPF grade animals after purchase Raise in the room, ensure sufficient food and light during the feeding period, and ensure suitable temperature and humidity.

[0171] Grouping of experimental mice

[0172] Before establishing the model mice, the included experimental mice should be divided into groups. 24 mice were selected and divided into PBS group, rCsHscB group, DDC group and (DDC+ rCsHscB) group according to the principle of random distribution. 6 mice. (Note: The rCsHscB mentioned in the full text refers to the recombinant protein CsHscB of Clonorchis sinensis).

[0173] A Modeling Approach for ...

Embodiment 3

[0210] Example 3: rCsHscB can inhibit the activation of hepatic stellate cells

[0211] 1. Cell culture of hepatic stellate cells LX-2

[0212] LX-2 cells were purchased from Xiangya School of Medicine, Wuhan University of Science and Technology. After routine resuscitation, the cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged, resuspended, counted, and inoculated on a 6 cm culture dish. The cells were divided into 4×10 5 / mL density inoculation. (6) Experiment grouping and cell processing:

[0213] 24 h after LX-2 adhered to the wall, PBS, TGF-β1 (15 ng / mL), TGF-β1 (15 ng / mL)+CsHscB (35 ug / mL), TGF-β1 (15 ng / mL) +RSG (1μM) was added to the culture dish, and DMEM was added to make up the volume to 2 ml. Placed at 37°C, 5% CO 2 Continue to grow in the incubator. After 24 hours, add 1 mL Trizol, mix well, and store at -80 °C for RNA extraction; add trypsin to digest, centrifuge, add PBS to wash, store at -80 °C for protein and total RNA ex...

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Abstract

The invention relates to the field of a biological technology, in particular to clonorchis sinensis recombination protein CsHscB (called rCsHscB for short) and an application thereof to cholestasis-induced liver fibrosis therapeutic drugs. The CsHscB protein has amino acid sequence shown as SEQ NO:2. Extensive and deep research finds that the protein has notable treatment effects on cholestasis-induced liver fibrosis. The protein treatment can obviously improve the liver function index and pathological changes of inflammatory cell infiltration, bile duct proliferation, cholestasis-induced liver fibrosis and the like of cholestasis-induced liver fibrosis animals. Besides, the recombination protein CsHscB can notably reduce the level of inflammatory factors (such as IL-6 and MCP-1 ) in cholestasis-induced liver fibrosis animal models. Therefore, the cholestasis-induced liver fibrosis therapeutic drug containing the rCsHscB protein provided by the invention has high application prospect and value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of Clonorchis sinensis recombinant protein CsHscB in medicine for treating cholestatic liver fibrosis. Background technique [0002] Cholestatic liver fibrosis is a type of cell injury and inflammation caused by stagnation of bile flow or failure of bile secretion and transport by hepatocytes and cholangiocytes, or by obstruction of extrahepatic free bile flow and excretion pathways, eventually leading to liver fibrosis. Unlike nonbiliary fibrosis, biliary fibrosis progresses rapidly and is accompanied by abnormal bile duct proliferation and peribiliary fibrosis. Clinically, the curative effect of drug therapy for intrahepatic cholestasis is not satisfactory. Although the current drug for the treatment of cholestatic liver fibrosis is ursodeoxycholic acid, the drug has strong toxic side effects and does not respond to some patients. In order to improve th...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P1/16C07K19/00C12N15/62
CPCA61K38/1767A61P1/16C07K14/43559C07K2319/21Y02A50/30
Inventor 颜超郑葵阳汤仁仙张钰于倩张雨钊武婧华慧李向阳范春阳张波
Owner XUZHOU MEDICAL UNIV
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